Sumoylation is essential for chromosome segregation; it is required for alignment of chromosomes at the metaphase plate, and for separation of sister chromatids at the metaphase to anaphase transition. Defects in these processes can lead to erroneous chromosome segregation, which is linked to development of human cancers and genetic diseases. The molecular mechanisms by which sumoylation controls progression through mitosis remain poorly understood. In particular, the proteins that are modified to affect control are largely unknown, and the mechanisms that regulate their spatial and temporal modification and demodification are also unclear. To address these issues, we will build on our recent discovery that the SUMO protease, SENP1, controls the progression from metaphase to anaphase by regulating inactivation of the spindle assembly checkpoint and/or activation of the anaphase promoting complex/cyclosome. The goals of our proposed studies are to define the molecular mechanisms by which sumoylation regulates anaphase onset and to determine how SENP1 affects precise spatial and temporal control of sumoylation in mitosis. Aims of the proposal include: (1) Defining the roles of SENP1 and sumoylation in regulating anaphase onset, (2) Defining the relationships between SENP1 localization, phosphorylation and mitotic function, and (3) Identifying SENP1 substrates and defining how localization and phosphorylation affect substrate selectivity.